利用电穿孔提高抗肿瘤药物疗效的研究
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摘要
细胞膜是电场作用的关键靶位点，它由液晶态和晶态两种状态的磷脂双分子层构成，具有一定的流动性和通透性。细胞膜对于一些小的分子是自由通过的，对大分子物质等则具有选择性，形成通透性屏障。细胞膜受到高强度脉冲电场处理时，其通透性障碍受到抑制，电穿孔就是形成短暂的通透性结构或膜上形成微孔。这种微孔能维持20多毫秒，最后因膜的流动性而闭合。不同的生物材料用相同的电场处理，穿孔的几率不同；同样，相同的生物材料用不同的电场处理，穿孔的几率也不同。本实验用天津理工学院生厂的基因脉冲导入仪处理Hela细胞，在不同的电场参数(电压，电容，脉冲次数)处理下，电穿孔的几率不同，从实验中找到了穿孔的最佳条件，在扫描电镜下观察到了电穿孔，电穿孔的深度与电压有关。Hela细胞受到电场处理时，表面的微绒毛受到不同程度的损伤。
利用电穿孔把抗肿瘤药物(环磷酰胺)导人Hela细胞，经台盼蓝染色和细胞流式仪测定，细胞的存活率大大降低，凋亡和死亡率大大增加，在小剂量时就能达到很大的胞质毒性。运用丫啶染色可以看到典型的凋亡细胞，核固缩、断裂。还发现，当药物达到一定的浓度时，电化学处理细胞的存活率与药物的浓度无关，即低剂量组与高剂量组的细胞存活率相等；在相同的药物浓度范围内，化疗组的细胞存活率明显与浓度有关；只用电场处理的细胞存活率与对照组相比发现，电场使细胞的存活率降低。
将电穿孔与环磷酰胺结合用于治疗KM小鼠S180肉瘤细胞异种移植肿瘤，电化学组的治愈率和有反应的比例都比只用电场和只用环磷酰胺(化疗)及对照组高。用电场处理肿瘤后，肿瘤部位不同程度的发生水肿，但随后水肿消失，表面结痂，肿瘤体积缩小直至消失。剥离未完全消退的电化学处理肿瘤与对照组肿瘤，比较发现，前者的表面几乎没有血管分布，对照组的血管非常丰富。
Cell membrane is the crucial target of electromagnetic pulse field (EMPF). In is composed of bilayer phosphate and protein including liquid crystal and solid crystal,and has the nature of fluidity and permeability. The permeability barrier is withheld when the cell membrane is exposed to a short high voltage EMPF. The electroporation is the transient pore or permeable structure on the cell membrane that will be last for 20 milliseconds and will be closed at the end because of the fluidity. The electroporation ratio of the different kinds of cell exposed to the same EMPF is different,so do the same cell exposed to the different EMPF. Furthermore,the viability of cell and the kind of buffers also affect the ratio of poration. In this paper,Hela cell handled by EMPF with various voltages,times,capacity,is contrast put in with unhandled cell,the handled cell is found to have been electropored. The frequency,pores number,depth of poration are relative to the EMPF parameters. At the same time,the microvilli are da
maged. Furthermore,the pore likes of volcanic vent and ejecta have been seen,then the best parameters of EMPF to Hela cell have been found. All above provide primary evidence for the electrochemotherapy of Hela cell.
CTX is delivered into Hela cell by electroporation. By cell flow sorter and the stains of typannan,the life ratio is reduced very much,the apoptosis ratio and the death ratio are increase severely,and the cytotoxicity at the low pose is so tense as the high pose. By the stain of yading,the quintessential apoptosis cell with nuclear condensity and the flaw have been founded. Furthermore,the life ratio has nothing to
do with the density of ami tumor drug when the density reaches some
degree.
As a result of EMPF influence,the life ratio of cell handled only by
EMPF is less low than the control cell' s.
The subcutaneous xenograft of slSO tumor cell on the KM rats has been used for experiments evaluating the response of tumors to various therapies. The cure ratio and the partial response ratio of the rats theraped by ECT is more high than the rats theraped by drugs only or EMPF only. The control rats have no response. Results show severe necrosis and edema in nearly all the rats treated by the drug and the pulse. The group with only drug treatment did not show any significant edema and the tumor volume of the control group was found to increase more much,but the tumor volume regressed with drug in combination with electric field.
Phlyctenes were observed at the sites of electrode contact with the skin,superficial leukonecrosis of some nodules was visible within the following 3 days,and scabs were observed a few days later,and the tumor volume decrease until regression. Separating the tumor from the body of rats. The tumor of rats treated by the drug and the pulse compared to the tumor of rats treated only with the drug or the pulse has little of vasculature,and the control has quite a few of vasculatures. Also,histopathological studies indicated that all the tumor cell was necrosis and the tissue from the tumor site showed complete absence of tumor cells when the tumor volume was complete regression . No tumor reappeared within 20 days.
利用电穿孔把抗肿瘤药物(环磷酰胺)导人Hela细胞，经台盼蓝染色和细胞流式仪测定，细胞的存活率大大降低，凋亡和死亡率大大增加，在小剂量时就能达到很大的胞质毒性。运用丫啶染色可以看到典型的凋亡细胞，核固缩、断裂。还发现，当药物达到一定的浓度时，电化学处理细胞的存活率与药物的浓度无关，即低剂量组与高剂量组的细胞存活率相等；在相同的药物浓度范围内，化疗组的细胞存活率明显与浓度有关；只用电场处理的细胞存活率与对照组相比发现，电场使细胞的存活率降低。
将电穿孔与环磷酰胺结合用于治疗KM小鼠S180肉瘤细胞异种移植肿瘤，电化学组的治愈率和有反应的比例都比只用电场和只用环磷酰胺(化疗)及对照组高。用电场处理肿瘤后，肿瘤部位不同程度的发生水肿，但随后水肿消失，表面结痂，肿瘤体积缩小直至消失。剥离未完全消退的电化学处理肿瘤与对照组肿瘤，比较发现，前者的表面几乎没有血管分布，对照组的血管非常丰富。
Cell membrane is the crucial target of electromagnetic pulse field (EMPF). In is composed of bilayer phosphate and protein including liquid crystal and solid crystal,and has the nature of fluidity and permeability. The permeability barrier is withheld when the cell membrane is exposed to a short high voltage EMPF. The electroporation is the transient pore or permeable structure on the cell membrane that will be last for 20 milliseconds and will be closed at the end because of the fluidity. The electroporation ratio of the different kinds of cell exposed to the same EMPF is different,so do the same cell exposed to the different EMPF. Furthermore,the viability of cell and the kind of buffers also affect the ratio of poration. In this paper,Hela cell handled by EMPF with various voltages,times,capacity,is contrast put in with unhandled cell,the handled cell is found to have been electropored. The frequency,pores number,depth of poration are relative to the EMPF parameters. At the same time,the microvilli are da
maged. Furthermore,the pore likes of volcanic vent and ejecta have been seen,then the best parameters of EMPF to Hela cell have been found. All above provide primary evidence for the electrochemotherapy of Hela cell.
CTX is delivered into Hela cell by electroporation. By cell flow sorter and the stains of typannan,the life ratio is reduced very much,the apoptosis ratio and the death ratio are increase severely,and the cytotoxicity at the low pose is so tense as the high pose. By the stain of yading,the quintessential apoptosis cell with nuclear condensity and the flaw have been founded. Furthermore,the life ratio has nothing to
do with the density of ami tumor drug when the density reaches some
degree.
As a result of EMPF influence,the life ratio of cell handled only by
EMPF is less low than the control cell' s.
The subcutaneous xenograft of slSO tumor cell on the KM rats has been used for experiments evaluating the response of tumors to various therapies. The cure ratio and the partial response ratio of the rats theraped by ECT is more high than the rats theraped by drugs only or EMPF only. The control rats have no response. Results show severe necrosis and edema in nearly all the rats treated by the drug and the pulse. The group with only drug treatment did not show any significant edema and the tumor volume of the control group was found to increase more much,but the tumor volume regressed with drug in combination with electric field.
Phlyctenes were observed at the sites of electrode contact with the skin,superficial leukonecrosis of some nodules was visible within the following 3 days,and scabs were observed a few days later,and the tumor volume decrease until regression. Separating the tumor from the body of rats. The tumor of rats treated by the drug and the pulse compared to the tumor of rats treated only with the drug or the pulse has little of vasculature,and the control has quite a few of vasculatures. Also,histopathological studies indicated that all the tumor cell was necrosis and the tissue from the tumor site showed complete absence of tumor cells when the tumor volume was complete regression . No tumor reappeared within 20 days.
引文
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[2]山道茂。生物热力学导论。高等教育出版社。1987。
[3]Freeman S. A, Wang M. A, and Weaver J.C. Theory of electroporation of planar bilayer membrane:predictions of aqueous area, change in capacitance, and pore-pore separation. Biophysical Journal. 1994.67(7):43-56
[4]刘长军，张弘，王保义。瞬态电磁脉冲对细胞跨膜电位的影响。四川大学学报(自然科学版)，1999，36(5)
[5]高体玉，冯军，细胞电化学进展。化学进展。1998。10(3)：305—311
[6]钱凯先，邵键忠，李亚南。细胞生物化学原理。浙江大学出版社，1999
[7]田心棣。人造双分子膜。高等教育出版社。1987。
[8]Gaoley. p. canmd Easterly CE. Cell membrane potentials induced during exposure to EM fields. Electro-and Magnetebiology. 1994.13(2) 159-163.
[9]Schwan H.P. Frequency selective propagation of extracellar electrical stimuli to intracellur compartments. Electro-and Magnetebiology 1994.13(2)159-165
[10]赵南明，浦摹明。细胞组分的原位电泳理论。清华大学学报。1983，27(4)：47—58
[11]gernardi. P. and D' inzeo. G.A nonlinear anaysis of the effects of transient electromagnetic fields on excitable membranes .IEEE
[27]汪和睦、李金慧，细胞电穿孔导入DNA的动态过程。微生物学报。1994，21(2)：67—71
[28]郑强、赵南明。中国学(B辑)，1988，8：826
[29]彭勇、王子淑、王小行。低强度孙态电磁场下动物细胞的电穿孔效应。四川大学学报(自然科学版)。2000，4。
[30]W.R. Adey, Biological effects of nonionizing radiation.Am. Chem. Soc. Washington. 1981,271-292.
[31]T.S. Tenforde. Bioelectromagnetics. 1989,10(13):215
[32]王保义，杨杰斌，郭庆功等，毫微秒电磁脉冲的生物效应实验研究和机理分析，中国科学(C辑)，1997，27(1)：36。
[33]Adair R.K. Constraints on biological effects of weak extremely-low-freqency electromagnectic fields. Physical Review A. 1991,43(2):1039-1048.
[34]李顺、黄卡玛、杨春等。弱强度微波与液相小分子基元反应体系相互作用中非热效应的机理初探。微波学报。1995，15(4)：345-353。
[35]Marino A.A. Different outcomes in biological experiments involving weak EMFs:is chaos a possible explanation ?American Physiological Society. 1995:R1013-1018.
[36]Sabelnikov A.G. and E.S. Cymbalyuk, Clotting of donour DNA during electroporation of prokaryotes. Bioelectrochem Bioenerg, 1990,24:313-321
[37]Mouneimne Y. et.P.F. Tosi, Y. Gazi and C. Nicolau. Electroinsertion of xeno-glycophorin into the red blood cell membrane. Biochem Biophys:Res, comm..1989,159:34-40
[38]M. Blank, Goodman, Bioelectromagnetics. 1997,18(12):111.
[39]汪和睦、汪军。生物化学与生物物理进展。1994，21(4)：323
[40]刘长军、王保义等，低强度快速电磁脉冲导致电穿孔的研究。科学通报1999，Vol44，(6)：1157—1161．
[41]Sower A.E.,Chang D.C. Guide to electroporation and electrofusion. Sanpiego;Academe Press, 1992:119.
[42]Zimmermann. Biochim. Biophys. Acta.,1982,694:227
[43]Potter H. et al.P.N.S. USA, 1984,81:7161-7165
[44]Benz R, and Zimmermmann. Relaxation studies on cell membranes and lipid bilayers in the high electric field range. Bioelectrochem. Bioenerg.,1980,1:723-739
[45]Zimmermann, P Scheurich, G Pilwant and R Benz, Cells with manipulated functions:New perspectives for cell biology. Medicine and Technolohy. Angew, Chem. Int. Educ. Eng. 1981,20:625-644
[46]杨孔、王子淑等。四川大学学报(自然科学学报)2001，Vol，38(5)：574-579
[47]H. E. Jacob, et al. Z. Allg. Mikrobiol. 1981, 21 (3) :225-233
[49]Gunter A. Hofmann. Electrochemotherapy:transition from laboratory to the Clinic. IEEE ENGNEERING IN MEDICINE AND BIOLOGY. Novamber/december124-130.1996
[48]汪和睦等，酵母整细胞及原生质体的最佳电穿孔条件。生物物理学报，1986，2(4)：364-371
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